The Studies On Depressant Effect Of Short-Hairpin RNA Of Bcl-xL Gene Expression In HepG-2 Cells

Objective: Liver cancer is one of the most frequent malignant tumor in China. Block of cell apoptosis is an important mechanism on tumorigenesis and growth. The apoptosis repressed gene Bcl-xL is the main member of Bcl-2 family. Recently, it has been found that Bcl-xL overexpressed in a variety of human cancers. Therefore, abrogation of Bcl-xL expression could be a potential therapeutic strategy against human cancers. We aimed to investigate the inhibitory action of short hairpin RNA (shRNA) targeting at the Bcl-xL gene expression in HepG-2 cells and to determine the effect of shRNA on the change of cell growth and cell apoptosis.Methods: A pair of reverse repeated sequence targeting Bcl-xL mRNA were contrived and synthesized, with 9 nucleotide sequences between them, and were directionally cloned to the Pgenesil-2, a plasmid vector to construct a recombinant expression plasmid pshRNA-Bcl-xL, and LipofectamineTM2000, a liposome was used to transfect the hepatocelluar carcinoma cell HepG-2 and stable transductants were selected with G418. Depressant effect of the pshRNA-Bcl-xL on the Bcl-xL mRNA and proteins were detected by the RT-PCR and flow cytometry methods, respectively. The change of cell growth was detected by MTT. Cell apoptosis and the expression of bcl-2 and bax were detected by flow cytometry.Results: Recombinant expression plasmid pshRNA-Bcl-xL could inhibit the expression of Bcl-xL gene of the hepatoma carcinoma cell (HCC) HepG-2 Compared with the group without transfecting, the expressions of Bcl-xL mRNA and proteins were apparently lower in the group with transfecting. The inhibition rates were 86.6% and 53.3% respectively. The cell growth became significantly slowly, and promoting apoptosis simultaneously. The expression of Bcl-2 was very low in HepG-2, and was't affected by shRNA that targeting at the Bcl-xL gene, but bax increased significantly.Conclusion: Construction of the recombinant expression vector pshRNA-Bcl-xL could significantly inhibit the expression of Bcl-xL gene, which results in the hepatocelluar cell apoptosis increasing, and the cell proliferation suppressing.